Journal of Immunological Methods 386 (2012) 6069

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

Research paper

A method for inducing antigen-specic IgG production by in

vitro immunization
Mieko Kato a, b, Huimin Yan a, Noriko M. Tsuji a, Tomoki Chiba b, Yoshiro Hanyu a,
a
Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),
1-1-1 Higashi, Tsukuba, 3058566, Japan
b
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 3058572, Japan

a r t i c l e i n f o a b s t r a c t

Article history: In vitro immunization (IVI) possesses a number of advantages over conventional immuniza-
Received 27 April 2012 tion. However, the number of positive clones derived from IVI is limited, and the affinity of the
Received in revised form 22 August 2012 antibodies from derived clones is relatively low. Moreover, the majority of immunoglobulins
Accepted 29 August 2012 produced in culture are IgMs instead of IgGs, which limits the application. Here, we report an
Available online 10 September 2012
improved protocol for IVI using mouse spleen cells. This protocol consists of multiple cycles of
repeated antigen stimulation followed by cell expansion, which increases the frequency of
Keywords: plasma cells that produce antigen-specific IgG antibodies. The culture conditions, including the
B cell cell density, the type of stimulants, and the initial cell preparation, were found to be important
CpG oligonucleotide
for inducing the IgG response. In addition, an analysis of the genes and cytokines expressed
Helper T cell
during the IVI showed that the antigen-specific B cells were specifically activated via
Hybridoma
IgG CD4-positive helper T cells. As evidence for this concept, our IVI protocol enabled us to
In vitro immunization establish an IgG antibody against keyhole limpet hemocyanin with a dissociation constant in the
order of 107 M.
2012 Elsevier B.V. All rights reserved.

1. Introduction
The fields of research, diagnostics, and therapeutics derive
significant benefits from antibodies. Thus, obtaining a good
antibody with both high affinity and selectivity to an antigen is
essential for meeting a variety of requirements in these
different fields. However, the ability to establish an antibody
with the desired affinity and selectivity against different types
of antigens remains challenging. In particular, difficulties
remain in making useful antibodies against certain antigens,
Abbreviations: IVI, in vitro immunization; CpG ODN, CpG oligodeoxy-
nucleotide; PEG, Polyethylene glycol; PBS, Phosphate buffered saline; FBS,
Fetal bovine serum; IL-2, Interleukin-2; IL-4, Interleukin-4; IL-5, Interleukin-
5; IL-10, Interleukin-10; IL-12, Interleukin-12; GM-CSF, Granulocyte macro-
phage colony-stimulating Factor; IFN-, Interferon-; TNF-, Tumor
necrosis factor-; TLR 9, Toll-like receptor 9; NK cell, Natural killer cell;
MDP, Muramyl dipeptide; ELISPOT, enzyme-linked immunospot; PBMCs,
peripheral blood mononuclear cells; Tr1, Type 1 Regulatory T cells.
Corresponding author. Tel.: +81 29 861 2716; fax: +81 29 861 2706.
E-mail address: y.hanyu@aist.go.jp (Y. Hanyu).
such as those that occur in small amounts, that are toxic, or that
show high homology to antigens of the host animals.
With in vitro immunization (IVI), immune cells isolated
from nave animals are stimulated with an antigen in vitro,
thereby resulting in the induction of B cells that produce
antigen-specific antibodies (Zafiropoulos et al., 1997). Sub-
sequently, such cells are fused with myeloma cells to form
hybridomas that produce the antigen-specific monoclonal
antibodies desired. IVI shows potential merit in the genera-
tion of many types of antigens, including those that are
problematic for in vivo immunization. That IVI is free from
the injection of animals with the antigen is an additional
advantage of this method in avoiding antigen toxicity
rendered failture. With further development, IVI should
especially be useful for obtaining human monoclonal anti-
bodies (Matsumoto et al., 2008). If one can establish human
monoclonal antibodies with IVI using human peripheral
blood mononuclear cells (PBMCs), complicated processes,
such as the humanization or chimerization of antibodies
(Kettleborough et al., 1991), could be avoided.

0022-1759/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jim.2012.08.019
 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069
To date, IVI methods have been established by treating the
PBMCs with L-leucyl-L-leucine methyl ester (Matsumoto et al.,
2006) and applying potent stimulators, including MDP, IL-2,
and IL-4. Nevertheless, IVI has not been used widely until now,
because its procedures are complicated and have yielded
unsatisfactory results. In particular, the majority of clones
from IVI produce IgMs, which show suboptimal affinities to be
broadly useful. Consequently, useful antibodies with high
affinity are rarely obtained with the conventional IVI protocols.
In addition, most protocols still fail to deliver sufficient stimuli
to antigen-specific B cells for their expansion and/or differen-
tiation into antibody-producing cells. Therefore, it has been
argued that an improved IVI protocol with conditions for
inducing class-switching (Geha et al., 2003) and affinity-
maturation, processes that are essential for obtaining antibody-
producing cells in vivo, would be a powerful tool for creating
useful monoclonal antibodies (Peled et al., 2008).
In this study, we developed an IVI protocol that effectively
activates the immune cells. By improving the cell preparation
methods and the culture conditions, as well as selecting the
most suitable immune stimulants, we succeeded in establishing
an efficient method for IVI. This protocol enables the induction
of antigen-specific IgG antibody production. An analysis of
the genes and cytokines expressed during the IVI showed
that the antigen-specific B cells were specifically activated via
CD4-positive helper T cells.
2. Materials and methods
2.1. Mice and IVI
Six-week-old female BALB/c mice were obtained from SLC
(Japan). The mice were sacrificed, and their spleens were
removed aseptically. The spleens were squeezed, and single-
cell suspensions were prepared. The cells were washed once in
RPMI-1640 (Sigma Aldrich, St. Louis, MO), and re-suspended in
10 mL of RPMI-1640 containing 10% fetal bovine serum (FBS).
The red blood cells and the granule cells were removed by
using Lympholyte-M (Cedarlane Laboratories, Canada).
CD8-positive T cells and natural killer cells (NK) cells were
removed from the splenocytes with negative selection methods
by using anti-CD8 antibody-coated magnet beads and anti-
CD49b antibody-coated magnet beads (Miltenyi Biotech, CA),
according to the manufacturer's instructions. For the IVI, the
cells were then washed twice and incubated individually at
37 C for 2 days in RPMI-1640 containing 20% FBS, with 10 g of
keyhole limpet hemocyanin (KLH, Thermo Fisher Scientific,
Waltham, MA) as the antigen, and the following stimulants:
0.25 M CpG ODN (5-tccatgacgttcctgacgtt-3, Hokkaido System
Science, Japan) and 0.25 M MDP (Sigma Aldrich). The
stimulated cells were collected by centrifugation and then
expanded in the culture media with IL-2 at 10 ng/mL, IL-4 at
2.5 ng/mL, and IL-21 at 10 ng/mL (PeproTech Inc., Rocky Hill,
NJ) for 2 days. For the secondary antigen stimulation, these
expanded cells were collected by centrifugation and stimulated
with 0.25 M KLH, 0.08 M CpG ODN, and 0.25 M MDP.
2.2. ELISA
A 96-well enzyme-linked immunosorbent assay (ELISA)
plate was coated with 50 L of 5 g/mL KLH per well. A
61
blocking solution (Blocking Reagent for ELISA; Roche) was
applied, and the plate was incubated for 2 h. The plate was
subsequently washed with PBS containing 0.05% Tween-20
(PBS-T). Afterwards, 50 L of PBS containing the supernatant of
stimulated splenocytes was added to each well. After the wells
were washed, alkaline-phosphatase-labeled anti-mouse IgG or
IgM antibody (Chemicon, MA) was added. The amount of the
antigen-specific antibody was measured using the alkaline-
phosphatase substrate kit (Sigma Aldrich), and the plates
were read using a microplate reader (Model 680; Bio-Rad
Laboratories) at a wavelength of 405 nm. All experiments were
conducted twice, and the average signal intensity was used in
the analysis. Total IgG and IgM levels in the culture superna-
tants of the stimulated splenocytes were determined using
ELISA with a mouse IgG ELISA quantitation kit and a mouse IgM
ELISA quantitation kit (BETHYL, Montgomery, TX), according to
the manufacturer's instructions.
2.3. ELISPOT assay
The frequency of B cell-producing antigen-specific IgG
was determined using an enzyme-linked immunospot
(ELISPOT) assay. Multiscreen HA filtration plates (Millipore,
Billerica, MA) were coated with KLH at a concentration of
10 g/mL (50 L/well) and incubated overnight at 4 C. The
plates were then blocked for 2 h at 37 C with RPMI 1640
containing 10% FBS. After the plates were washed with PBS,
cells were added to the plates at a density of 5 105 cells/well.
The cells were cultured for 24 h at 37 C and 5% CO2. After the
culture period, the plates were washed with PBS containing
0.05% Tween 20 (PBS-T) and incubated with diluted goat
anti-mouse IgG antibody conjugated with alkaline phosphatase
(Southern Biotech, Birmingham, AL) for 2 h at 37 C. After the
plates were washed with PBS-T, Sigma Fast BCIP/NBT solution
(Sigma) was added, and the plates were incubated at room
temperature for 10 min. When the development was complete,
the number of spots was scored.
2.4. Cytokine measurements
Cytokine levels in the culture supernatants of stimulated
splenocytes were measured. Murine IL-2, IL-4, IL-5, IL-10,
IL-12, GM-CSF, IFN-, and TNF- levels were measured using
a Bio-Plex assay with a mouse cytokine group 1 Th1/Th2
assay kit (Bio-Rad Laboratories, Minneapolis, MN,) according to
the manufacturer's instructions.
2.5. Flow cytometric analysis
Splenocytes were restimulated with PMA (50 ng/ml;
Sigma-Aldrich) and Ionomycin (200 ng/ml; Sigma-Aldrich) in
the presence of GolgiStop. After staining with PE-Cy7-
conjugated anti-CD4 mAb, cells were fixed and permeabilized
with the Cytofix/Cytoperm Plus Fixation/Permeabilization Kit
(BD Biosciences). Intracellular cytokines were stained with
PE-conjugated anti-IL-4, APC-conjugated anti-IL-10, and FITC-
conjugated anti-IFN- mAbs. Flow cytometry was performed
using FACSAria (BD Biosciences), and data were analyzed using
FlowJo software (Tree Star, Ashland, OR).
62 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069
2.6. Gene expression
After RNA from stimulated cells was purified, the
corresponding cDNA was synthesized with TAKARA reverse
transcriptase. Using this cDNA as a template, real-time PCR
was carried out with a Terminal Cycle Dice TP800 (Takara Bio
Inc., Japan) using SYBR Green for the detection of the PCR
products. The reaction mixture (RT-PCR kit, Takara Bio)
contained 12.5 L of SYBR Premix Ex Taq (2 ) (Takara Bio),
10 M PCR forward primer (0.5 L), 10 M PCR reverse
primer (0.5 L), and cDNA (2 L) to give a final reaction
volume of 25 L. The sequences of the primers for Blimp-1, AID,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
were obtained using the Perfect Real Time support system
(http://www.takara-bio.co.jp/prt/imtro.htm). GAPDH expres-
sion was used for the normalization.
2.7. Establishment of monoclonal antibodies by IVI
For establishing mouse monoclonal antibodies against KLH
by IVI, splenocytes from BALB/c mice immunized with KLH in
vitro were isolated and mixed with an equal number of
myeloma cells. Murine myeloma cells P3X63Ag8U.1 (P3-U1)
were used as the fusion partners for the murine B cells. Cells
were routinely cultured in RPMI-1640 supplemented with 10%
FBS at concentrations between 1 105 and 1 106 cells/mL.
The 2 cell types were counted using a hemocytometer and
mixed together in a ratio of 1:1 for fusion. The cells were fused
by PEG methods. Next, the cell suspensions were pipetted into
10 mL of RPMI-1640 medium (free of phenol red) containing
20% FBS and 5% Briclone (QED Bioscience Inc, San Diego, CA).
The fused cells were suspended in a 96-well plate and grown at
37 C under a 5% CO2-enriched atmosphere. After 24 h,
hypoxanthine (HAT; Sigma) medium was added to each well.
The hybridomas generated were incubated in a 96-well plate
for 7 days. The supernatants from the individual wells were
screened by ELISA. The hybridomas that produced the
monoclonal antibodies were cloned by a limiting dilution
method, and the secreted antibody was purified with a protein
G column (GE Healthcare, MA, USA).
2.8. Kinetic analysis by surface plasmon resonance
Kinetic analysis was performed using a Biacore J system (GE
Healthcare). The KLH was amine coupled to the CM5 sensor chip
as instructed by the manufacturer (NHS/EDC coupling kit, GE
Healthcare). Purified monoclonal antibodies were dissolved in
HBS-EP buffer (0.01 M HEPES [pH 7.4], 0.15 M NaCl, 3 mM
EDTA, 0.005% surfactant P20) and injected as the analyte
solution. All measurements were performed at a flow rate of
30 L/min at 25 C, and the interaction surface was regenerated
with glycine-HCl (pH 1.5). Data were evaluated using the
Biaevaluation software (version 4.1; GE Healthcare).
3. Results
3.1. Procedure for IVI and total IgG and IgM production
Fig. 1 shows the experimental procedure used for inducing
the production of antigen-specific IgG antibodies in vitro. First,
splenocytes isolated from 6-week-old BALB/c mice were
treated to eliminate cytotoxic lymphocytes, such as CD8-
positive T cells and NK cells, through negative selection with
antibody-coated magnetic beads. Next, the first antigen
stimulation, which used CpG ODN and MDP as stimulants,
was applied to these cells for 2 days. Afterwards, the stimulat-
ed cells with the larger diameters were collected by low speed
centrifugation and then expanded with a cocktail of cytokines
for 2 days to activate IgG-producing cells. Antigen stimulation
was subsequently applied again for 2 days to induce the
production of the antigen-specific IgG. This 3-step activation
procedure enabled the efficient induction of antigen-specific
IgG-producing cells.
We measured the production of total IgG and IgM
production from the stimulated splenocytes during the IVI
(Fig. 2). Neither IgG nor IgM was produced without the
stimulation. In contrast, a single antigen stimulation evoked
the production of both immunoglobulins. Moreover, expansion
with either the cocktail of interleukins or the 2 successive
antigen stimulations increased the production of IgG more
than that of a single antigen stimulation, and induced the
highest production of IgM. Antigen-stimulated splenocytes
that had undergone expansion and the second antigen
stimulation showed the highest production of IgG but showed
decreased IgM production. The 3-step stimulation induced the
highest production of IgG but reduced the production of IgM
compared to the single and 2-step stimulations.
3.2. The induction of antigen-specic IgG antibody by in vitro
immunization
After the stimulation, the number of antigen-specific
IgG-producing cells among the activated splenocytes was
determined with ELISPOT (Fig. 3A). Antigen-specific IgG-
producing cells could not be induced by a single stimulation
with the antigen. The induction of the antigen-specific
IgG-producing cells occurred only upon the expansion. Further,
the 2nd stimulation after the expansion of the antigen-
stimulated splenocytes induced more antigen-specific IgG-
producing cells. The number of positive cells increased more
than 3-fold by the 2nd stimulation. The size and color of the
spots on the cells after the expansion were smaller and lighter,
respectively, than those of the spots on the cells after the 2nd
stimulation. The spots of the cells after the 3-step stimulation
were large and dark in color, indicating that antibodies were
being produced in large amounts and that the affinities against
the antigen were higher than those of the previous steps. Thus,
the only splenocytes after the 3-step stimulation produced
higher affinity antigen-specific IgG antibodies. The repeated
antigen stimulations did not increase antigen-specific IgG-
producing cells. Further, the employment of an expansion step
between the antigen stimulations appeared to be critical for the
production of antigen-specific IgG. Omitting any one of these 3
steps either substantially reduced or occasionally diminished
the production of antigen-specific IgG. Expansion after the 2nd
stimulation led to substantial cell death during culture (data
not shown). Thus, the second expansion is not effective for the
induction of antigen-specific IgG-producing cells.
Next, we examined the effects by the cell density during
the IVI by increase seeding density from 5 10 5 cells/mL to
1 10 7 cells/mL. As shown by ELISPOT, the number of cells
producing antigen-specific IgG antibody increased from
 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069
Splenocytes from The first antigen stimulation
BALB/c mouse
Culture for two days
Antigen, CpG ODN
and MDP
Fig. 1. Procedure for in vitro immunization (IVI). Isolated splenocytes were activated with a 3-step stimulation. After each stimulation, the activated vital cells
were collected by centrifugation. Afterward, the cells were suspended in fresh media, and the next stimulation was applied. CpG ODN, CpG oligodeoxynucleotide;
IL: interleukin; MDP, muramyl dipeptide.
1 10 6 cells/mL to 8 10 6 cells/mL (Fig. 3B). The number of
spots likewise increased with this increasing cell density.
The number of positive spots at cell densities of less than
2 10 6 cells/mL was very low. Meanwhile, the number of spots
at cell densities of 4 106 cells/mL were greater than 5 times
that at cell densities of 2 10 6 cells/mL. The maximum number
of spots were observed at cell densities of 8 10 6 cells/mL.
These results show that cell density has significant impact on
the induction of antigen-specific IgG and thereby indicate the
importance of cell-cell interactions during the incubation.
Fig. 3B shows the effect of CD4-positive T cells on the
production of antigen-specific IgG. When the CD4-positive
T cells were eliminated prior to the IVI, the production of
antigen-specific IgG remained at the basal level, indicating that
antigen-specific IgG production is dependent on the interaction
with CD4-positive helper T cells.
3.3. Gene expression during IVI
The expression of genes related to B-cell activation was
studied during the in vitro stimulation by real-time PCR (Fig. 4).
* nocytes during IVI to study activation signaling in our system
g / m L
1 st
Exp
2 nd
Fig. 2. Total IgG and IgM production during IVI. The concentration of total IgG
and IgM in the culture medium after various combinations of stimulation steps
were measured with ELISA. 1st denotes the concentration after the first
antigen stimulation; Exp, the concentration after the expansion with the
cocktail of cytokines; and 2nd, the concentration after the second antigen
stimulation. The columns represent the average concentration from 3
independent experiments. The error bars represent the standard deviation
(SD). *, pb 0.05 for stimulated vs. unstimulated cells.
63
Expansion The second antigen stimulation
Culture for two days Culture for two days
IL-2, IL-4,
IL 4, and IL-21 Antigen, CpG ODN
and MDP
The expression of Blimp-1 showed no increase as the culture
density changed from 1 106 cells/mL to 2 106 cells/mL.
However, its expression significantly increased as the cell
density increased from 4 106 cells/mL to 8 10 6 cells/mL.
Meanwhile, the expression of AID increased as the cell density
increased from 1 106 cells/mL to 8 106 cells/mL. The
expression of AID, which induces hypermutation in the
antibody gene and leads to affinity maturation, was ~80 and
~200 times higher at 4 10 6 and 8 106 cells/mL, respectively,
than that of nave splenocytes. The expression of Blimp-1 at
4 10 6 and 8 10 6 cells/mL was approximately 5- and
14-folder higher, respectively, than that of nave splenocytes.
The expression of these genes was known to correlate with
antigen-specific antibody expression. The expression of
Blimp-1 and AID showed that the B cells in this system were
activated through a similar activation pathway as in vivo.
Moreover, their expression induced the differentiation of B
cells into plasma cells during IVI and increased the affinity of
the antibody.
3.4. Cytokine expression during IVI
We measured the cytokine expression profile of sple-
(Fig. 5). Three expression pattern profiles were observed. The
production of IL-2 and IL-10 was induced by antigen stimula-
tion but minimally or not at all by the expansion step.
Meanwhile, IL-12 and TNF- production were induced by the
expansion step but only slightly by antigen stimulation. The
stimulants in our system may have directly stimulated the
splenocytes and caused them to produce these cytokines.
However, IL-4, IL-5, IFN-, and GM-CSF showed a remarkable
increase in expression only after the 3-step stimulation. These
cytokines were not produced by either the single antigen
stimulation, the single expansion step, or their combination.
Hence, the 3-step stimulation was critical for the production of
these cytokines. Thus, 3 successive stimulations induced cell
population changes important for this response. Among these
cytokines, IL-4 and IL-5 are produced from Th2 cells, while
IFN- and GM-CSF are produced from Th1 cells. IL-10, which
showed increased expression upon antigen stimulation, was
produced from Th2 (IL-4- and IL-10-producing) and Tr1
(IL-10-producing and IL-4-non-producing) cells. IL-12, which
displayed increased expression during the expansion step, is
produced from accessory cells, such as macrophages, and
eventually enhances IFN- in Th1 cells. Thus CD4-positive
helper T cells were effectively stimulated in our system and
64 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069

A B
* *
*
Spots / 5x105

Spots / 1xx106
* * * *

1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL 4x106/mL

1st - + + + + +
Splenocyte CD4-
Exp - - + - + -
2nd - - - + + +
3rd - - - - - +

Fig. 3. Induction of antigen-specific IgG-producing cells in IVI. A. The number of antigen-specific IgG-producing cells was counted by ELISPOT after various
combinations of stimulation steps and cell densities. 1st, Exp, and 2nd denote the same steps as in Fig. 2. 3rd denotes the third antigen stimulation. The
fusion efficiency was determined as in B. CD4 denotes the experiment in which CD4-positive cells were removed by negative selection from the splenocytes
prior to stimulation. The columns represent the average concentration from 3 independent experiments. The error bars represent the standard deviation (SD).
*, p b 0.05 for stimulated vs. unstimulated cells.

differentiated into Th1 and Th2 cells to produce the cytokines,
which presumably work in concert to promote IgG production.
3.5. Role of helper T cells in the immunization
As shown above, the CD4-positive helper T cell is
indispensable for the induction of antigen-specific IgG
antibodies for this in vitro system. Hence, the CD4-positive
helper T cells from splenocytes stimulated at various cell
densities were characterized. The intracellular cytokines IL-4,
IL-10, and IFN- were stained with fluorescein-labeled
antibodies and measured by flow cytometry. The helper T cells
were stained using anti-CD4 antibody. A dot analysis of the flow
cytometry values for cultures at a density of 4106 cells/mL is
shown in Fig. 6A. The ratio of Th1 cells (IFN--producing and
IL-4-non-producing) to CD4-positive helper T cells decreased
with increasing cell density. The maximum ratio was 7.5% at a
cell density of 2 106 cells/mL (Fig. 6B). However, for the Th2
cells (IL-4-producing and IFN--non-producing), the ratio
increased with the cell density and showed a maximum
(7.5%) at a density of 4 10 6 cells/mL. The ratio of IL-4 and
IL-10-producing CD4-positive helper T cells to IL-4-producing
CD4-positive helper T cells increased with increasing cell density
(Fig. 6B), showing a maximum value (26.2%) at a cell density of

Blimp1 AID
16
* 250

*
Gene expression

Nave 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL Nave 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL

Fig. 4. Expression of genes after IVI. Expression of Blimp-1 and AID was measured by real-time polymerase chain reaction analysis. Further, glyceraldehyde-
3-phosphate dehydrogenase expression in nave splenocytes was used for the normalization. The error bars represent the SD. *, p b 0.05 for stimulated vs.
unstimulated cells.
 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069 65

IL-2 IL-12 IFN- TNF-

L
pg/mL

IL-4 IL-5 IL-10 GM-CSF

pg/mL

1st + + + + + + + + + + + +
Exp - + + - + + - + + - + +
2nd - - + - - + - - + - - +
Fig. 5. Cytokine production during in vitro immunization. Splenocytes were activated with various combinations of stimulation steps. The supernatants were
collected, and cytokine production (as indicated by IL-2, IL-4, IL-5, IL-10, IL-12, tumor necrosis factor [TNF6-, interferon [IFN]-, and granulocyte macrophage
colony-stimulating factor [GM-CSF]] was evaluated by Bio-Plex. 1st, Exp, and 2nd denote the same steps as in Fig. 2. The data represent the mean of 3
replicates. The error bars represent the SD.

A
105

105
104

104
IL--4

103

103
102

102

102 103 104 105 102 103 104 105

IFN-
- IL-10
-
B
Th1(IFN- +, IL-4 -) / CD4 + Th2 (IL-4 +, IFN-) / CD4 + IL-10
- + /IL-4 +
+ cells %

4+ cells %

+ cells %
CD4+

IL--4+, IFN-- cells / CD4

+ cells / IL-4+
IFN-- +, IL-4 cells / C

L-10+
IL

1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL

Fig. 6. Intracellular staining of cytokines in CD4-positive cells in IVI. A. FACS profiles from intracellular cytokine staining for a culture density of 4 106 cells/mL is
shown. B. The ratio of cells producing each cytokine to CD4-positive cells was measured with the changing cell density. The columns represent the values from 3
independent experiments. The error bars represent the SD.
66 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069
8106 cells/mL. Thus, Th2 cells were preferentially produced
when the cell density was above 4106 cells/mL; Th1 cells,
when the cell density was low.
To study the effect of CD4-positive helper T cells on signal
transduction during IVI, we measured cytokine production
with and without CD4-positive helper T cells at a cell density
of 4 10 6 cells/mL (Fig. 7). The production of IL-4, IL-5, IL-10,
IL-12, IFN-, TNF-, and GM-CSF was dramatically reduced
when the CD4-positive helper T cells were depleted. By
contrast, IL-2 production was increased with their absence.
These results show that these different cytokines, with the
exception of IL-2, are produced either directly from CD4-positive
helper T cells or from cells activated by them.
3.6. Establishment of an monoclonal antibody with IVI
Next, we attempted to establish a monoclonal antibody by
using our IVI method. After immunization with KLH, 5 10 7
splenocytes were fused with 2.5 108 myelomas. IVI and
fusions were independently performed. An average of 356
94 (n= 3) hybridoma colonies were obtained from each fusion
experiment. Among these colonies, an average of 3.3 1.1
(n= 3) clones produced the antigen-specific IgG antibody,
while an average of 12.7 4.1 (n= 3) clones produced the
antigen-specific IgM antibody. The ratio of antigen-specific
IgG-producing clones to antigen-specific IgM-producing clones
was 3.8. Positive hybridomas were cloned by the limiting
dilution method. The affinity of the purified anti-KLH mono-
clonal IgG antibody was measured by Biacore (Fig. 8). The
monoclonal antibody was shown to bind to the antigen in a
concentration-dependent manner. The KD of the antibody was
calculated as 5.21 107 M, with an association rate constant
of 1.47 10 5 M1 s1 and a dissociation rate constant of
7.66 102 s1. Hence, our in vitro method could be used to
obtain a monoclonal antigen-specific IgG antibody.
4. Discussion
IVI possesses a number of advantages over conventional
immunization. However, its low efficiency in B-cell activation
500
300
pg/mL
Fig. 7. Cytokine production after IVI. The effect of CD4-positive cells on cytokine production was analyzed by Bio-Plex. CD4 positive-cells were removed from the
splenocytes prior to the stimulation. The culture density was 4 106 cells/mL. The data represent the mean of 3 replicates. The error bars represent the SD.
40
35
30
3.07 M
Response (RU)
KD=5.21 10--7 (M)
25
20
15
1.537 M
0.769 M
10
5
0
0 50 100 150 200
Time (sec)
Fig. 8. Kinetic analysis of the binding of keyhole limpet hemocyanin (KLH) to
the monoclonal antibody by using the Biacore system. The purified antibody
was immobilized onto a CM5 sensor tip. KLH was injected over the biosensor
surface twice at the indicated dilutions. The flow rate was 30 L/min. The
experimental curves (thin lines) were fitted globally (thick lines) using
the mass transfer-limiting model. KLH was injected at concentrations of
3.07 nM, 1.537 nM, and 0.769 nM.
limits the number of positive cells and the insufficient matu-
ration of antibody-producing cells. This difficulty results from
the lack of effective immune stimulation and is inherent to the
procedure, which involves mixing cells, an antigen, and some
immune stimulants (Zafiropoulos et al., 1997). Increasing the
number of positive clones is a crucially important step in
obtaining the desired antibody. Moreover, establishing an
effective IVI protocol for inducing cells that produce antigen-
specific IgG antibodies with a high affinity is essential. Stim-
ulating B cells to differentiate into plasma cells is necessary for
obtaining IgG antibodies with high affinity and thus imperative
for an effective IVI protocol. For this purpose, MDP, LPS, PWM,
IL-2, and anti-CD40 antibody (Chin et al., 1995; Saito et al., 2008;
Tamura et al., 2007) have been used as immunomodulators
during IVI. However, the effects of these stimulators are still
restrictive and insufficient, and methods employing these
stimulators tend to require relatively long stimulation periods,
such as 19 days for PMBCs (Chin et al., 1995). As a result,
considerable cell death tends to occur. Schilizzi et al. (1992)
6000
5000
3000
 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069
added T cell clones to the culture and succeeded in obtaining
antigen-specific IgG, but this method is only applicable when T
cell clones against the antigen do not already exist.
Here, we report an efficient procedure for IVI. We have
established procedures for the cell preparations and culture
and succeeded in the induction of antigen-specific IgG antibody
production. Our procedure consists of 3 steps for inducing
antibody production in splenocytes: 1) an initial antigen
stimulation, 2) cell expansion with a cocktail of cytokines,
and 3) a second antigen stimulation. With our procedure,
antigen-specific IgG-producing cells can be obtained in the
short period of 6 days. The ELISPOT analysis showed that the
3-step stimulation not only induced antigen-specific IgG
antibodies but also facilitated the affinity maturation of
resultant antibodies. The order of these steps appears to be
important for obtaining good results. For the first antigen
stimulation, CpG ODN (Bal et al., 2011; Kato et al., 2011) is
applied with the antigen and effectively stimulates the B cells,
especially the antibody-producing ones. In the expansion step,
the T and B cells are stimulated directly. This expansion causes
cell proliferation, with the resultant increase in cell density
potentially strengthening cell-cell interactions (Haniuda et al.,
2011; Nojima et al., 2011). This step is essential for the increase
of antigen-specific IgG-producing cells at the end of the culture
(Fig. 3A). When the second antigen stimulation was applied to
the antigen-stimulated splenocytes following the expansion
step, the number of antigen-specific IgG-producing cells
increased more than 3-fold. However, this increase with the
second antigen stimulation was not observed if the expansion
step was omitted (Fig. 3A). The second antigen stimulation
activates the antigen-specific antibody-producing cells and
may effectively induce IgG production. An expansion after the
second antigen stimulation also increased the ratio of positive
cells, but this increase was not effective for the efficient
induction of antigen-specific IgG-producing cells, since the
total number of splenocytes was decreased drastically after the
second expansion. This loss of cells during culture decreased
the total number of positive cells. When CD8-positive helper T
cells and NK cells were not removed prior to the IVI, the cell
death worsened, and the total cell number decreased
(Zafiropoulos et al., 1997). The production of antigen-specific
IgG antibodies was not observed with this preparation. Thus,
the removal of the cytotoxic lymphocytes was indispensable
for the induction of antigen-specific IgG production.
The highest production of total IgG in our experiments so
far was induced by the 3-step stimulation (Fig. 2). The single
antigen stimulation resulted in IgG production that was 40%
of that of the 3-step stimulation. The second antigen stimu-
lation after the expansion increased the production of total
IgG but reduced that of IgM. This expression pattern of total
IgG and IgM indicate that class switching occurred after the
second antigen stimulation. However, antigen-specific IgG
was not produced by a single antigen stimulation in our
procedure (Fig. 3A). By contrast, antigen-specific IgG pro-
duction was remarkably high after the 3-step stimulation,
with the third step showing a marked increase in production.
The pattern of production during the procedure was different
between total IgG and antigen-specific IgG. The production of
total IgG showed little change between the expansion and
the second antigen steps. Hence, the third stimulation is
specific to the antigen-specific IgG-producing cells. These
67
results show that our system worked well for inducing an
antigen-specific response. While this has never been ex-
plored before in an IVI protocol, we directly examined the
presence of cells producing antigen-specific IgG antibodies by
ELISPOT. Zafiropoulos et al. (1997) estimated the number of
cells producing antigen-specific IgG antibody indirectly from
ELISA methods. Tamura et al. (2007) also induced the
antibody from PMBCs and counted the number of antigen-
specific antibody-producing cells by ELISPOT. However, these
researchers counted the number of cells producing IgM but
not IgG. Total IgG production was not increased over IgM
production in their IVI method, which shows that class
switching had occurred but not efficiently.
Cell density was one of the important factors for the
induction of antigen-specific IgG antibodies. Antigen-specific
IgG antibodies were effectively induced at a cell density
greater than 4 10 6 cells/mL, while no antigen-specific IgG
antibody was induced at a density less than 2 10 6 cells/mL.
However, this high density was not good for the cell culture.
To improve the culture conditions, we collected the activated
cells with low centrifugation after each stimulation and
restarted the culture at a fixed cell density. This procedure
enabled the selection of activated vial cells and resulted in a
cell culture with better conditions.
The importance of cell density in the culture was further
supported by 2 observations made during the course of this
study. First, as in the vivo immune response, interactions
between CD4-positive cells and B cells (Kouyama et al., 2007;
Okazawa et al., 2008) were crucial for inducing antigen-
specific IgG in the IVI system. When the CD4-positive helper
T cells were eliminated from the system, cytokine production
was suppressed, and antigen-specific IgG production was not
induced. Thus, our results indicate that the in vivo in-
teractions responsible for IgG induction were at least partly
reproduced in our in vitro system. Moreover, the increase in
the production of antigen-specific IgG antibody with increas-
ing cell density (Fig. 3) suggests that the interactions
between CD4-positive cells and B cells were strengthened
in the high density cultures. Second, real-time PCR analysis
showed that Blimp-1 (Nutt et al., 2007) and AID (Pavri and
Nussenzweig, 2011) were expressed during the IVI, indicat-
ing that the B cells in our system are activated in the same
way as in vivo. Further, the expression of AID showed that
somatic hypermutation (Cumbers et al., 2002) and class
switching (Stavnezer et al., 2008) had occurred during the
IVI. The expression of Blimp-1 shows that the B cells
differentiated into plasma cells (Diehl et al., 2008; Nutt et
al., 2011). The expression of these genes was markedly
increased when the cell density of the culture was high. The
increased expression of these genes correlated well with an
increase in antigen-specific antibody expression.
The cytokine milieu is another critical factor for successful
IVI. After the 3-step stimulation, the production of cytokines,
such as IL-4, IL-5, IL-10, GM-CSF, and IFN-, was increased.
These cytokines activate the antibody-producing cells and
induce IgG production, class switching, and affinity matura-
tion. These cytokines would have the effect of increasing and
decreasing the production of total IgG and IgM, respectively,
after the 3-step stimulation (Fig. 2). IFN- and GM-CSF
activate macrophages and induce colony formation (Metcalf
et al., 1986) and MHC-II expression (Giroux et al., 2003),
68 M. Kato et al. / Journal of Immunological Methods 386 (2012) 6069
respectively. This would increase the antigen presentation to
the CD4-positive helper cells and induce the differentiation
and proliferation of antibody-producing cells. The cytokines
produced after the 3-step stimulation are from both Th1 and
Th2 cells. Hence, helper T cells in our system differentiated
into both Th1 and Th2 cells (Anderson et al., 2007; Murphy
and Reiner, 2002), which are involved in cell-mediated
immunity and humoral immunity, respectively. The Th2
cells induced the differentiation of B cells into plasma cells,
which produced the antigen-specific antibody. The differen-
tiation of helper T cells into Th2 cells is desirable for the
induction of antigen-specific IgG, but differentiation into Th1
was also observed in the system. Hence, further potent
stimulation should be applied to differentiate helper T cells
into Th2 cells.
Intracellular staining of the cytokines showed that IL-4 and
IL-10 production from CD4-positive helper T cells, which
activate antibody production by B cells, was greater at the
higher cell densities. These results show that Th2 is predom-
inant when the cell density is high and that higher cell densities
are thus preferable to antigen-specific IgG production, again
explaining why cell densities above 4 106 cells/mL resulted in
the excellent formation of spots representing the production of
antigen-specific antibodies. Th1 is predominant when the cell
density is low during IVI. In our protocol, we used the cell
density at 4 106 cells/mL for IVI because these conditions
resulted in the predominance of Th2 and were thus suitable for
the production of antigen-specific IgG antibody.
We succeeded in establishing an IgG monoclonal anti-
body against KLH with IVI. One of these clones was
subcloned, and the monoclonal antibody was purified and
shown to have a KD of 5.21 10 7 M, with an association
rate constant of 1.47 10 5 M 1 s 1 and a dissociation rate
constant of 7.66102 s1. Hence, obtaining a monoclonal
antigen-specific IgG antibody is possible with our in vitro
method. However, the KD of the antibody was not as high as the
one from a normal in vivo method (Sasamori et al., 2011). Chin
et al. (1995) established a monoclonal antibody from PMBCs
using IVI. This monoclonal antibody had a KD of 2.4108 M,
with an association rate constant of 8.5103 M1 s1 and a
dissociation rate constant of 2.0104 s1. The association rate
constant of our antibody was less than that of their antibody by
approximately 2 orders of magnitude. Conversely, the dissoci-
ation rate constant of our antibody was greater by approxi-
mately the same order of magnitude. One worthy future
experiment is to collect more clones to examine whether higher
affinity clones are in fact present and obtainable by IVI. The
combination of short experimental cycle of 6 days relative to
19 days of conventional methods and the ability to handle
highly toxic antigens makes IVI highly attractive. The other
monoclonal antibodies from our IVI have the same character-
istics, with smaller association rate constants and larger
dissociation rate constants. This would be related to the affinity
maturation process in our IVI. Affinity maturation that results in
a reduced dissociation of the antibody from the antigen would
be necessary in our system for the further improvement of
affinity. The results show that our in vitro method can induce a
sufficient number of B cells to produce IgG-antibody specific to
the antigen. For the IVI, the ELISPOT analysis showed that the
ratio of positive clones was in the order of 105. Zafiropoulos et
al. (1997) induced monoclonal IgG antibody from PMBCs with
IVI and showed that the ratio of positive clones was in the order
of 106. Hence, the positive ratio from our IVI method was 1
order of magnitude higher. Further, 1012 days were needed
for their immunization, while only 6 days were required for our
method. The number of antibody-producing cells is also
important for establishing hybridomas. To establish a hybrid-
oma, the ratio of positive cells should be high, since the fusion
rate is as high as 104. If we use 108 cells for the fusion, the ratio
of positive cells should be higher than 105 to obtain enough
positive cells. This value was not sufficient for obtaining enough
hybridomas after the cell fusion. In our experiment, we obtained
an average of 3.3 clones from 1 fusion. This number was not
sufficient to obtain antibodies with high affinities, selectivity, or
functional modulations.
Nonetheless, our results also show either that the affinities
of these antibodies have not been sufficiently increased for
practical use or that the number of B cells producing an
IgG antibody with a high affinity remains small. For further
improving the number of positive clones and IgG antibody
affinity of IVI, more efficient activation is required. For this
purpose, culture conditions that are closer to the in vivo
situation should be applied to the splenocytes. In essence, the
conditions in the germinal center (Victora and Nussenzweig,
2012) should be reproduced in vitro during immunization. The
co-culture of splenocytes with follicular dendritic cells
(Nishikawa et al., 2006) and T helper cell clones (Uthoff and
Boldicke, 1993) are likely to activate B cells more efficiently.
These stimulations would induce class switching and affinity
maturation more effectively.
5. Conclusion
Our method not only induces total IgG production but also
produces an antigen-specific IgG relative to other methods.
ELISPOT analysis showed that our 3-step stimulation proce-
dure increased the number of antigen-specific IgG-producing
cells and induced antigen-specific IgG antibody production.
This procedure induced the expression of B cell activators
including Blimp-1 and AID; and the activation of cytokine-
producing CD4-positive helper cells. The expression pattern
of total IgG and IgM indicate that class switching occurred
after the second antigen stimulation. Thus, the 3-step stimula-
tion appears to mimic important processes that occur in vivo.
The application of our method is expected to increase the
probability of obtaining a desired antibody against a challeng-
ing antigen. With improved efficiency, this method is also
expected to be applicable to the production of human mono-
clonal antibodies.
Acknowledgement
This work was partially supported by the Programme for
Promotion of Basic and Applied Researches for Innovations in
Bio-oriented Industry.
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